1. Field of the Invention
The invention relates to a particular type of β-glucan—a microbial polysaccharide composed of D-glucose in either straight or branched chains with glycosidic linkages. More specifically, the present invention is directed to industrial and commercial applications of a β-1,3-1,6-glucan appearing in the medium of the black yeast, Aureobasidium. Aureobasidium is one of several genera of black yeasts, characterized by mostly slow-growing, black, pasty colonies. β-1,3-1,6-glucans are complex acidic polysaccharides having a main chain comprising subunits having 1,3 linkages and branches at the 3- and 6-positions. The β-glucans of the present invention have a variety of industrial and commercial uses, including applications in pharmaceutical or medical products or treatments, for the removal or control of environmental or microbiological contaminants, in cosmetics, and in nutritional products and foods.
2. Description of the Related Art
Various microbial and fungal polysaccharides with different structures and diverse physiological properties are known. Such polysaccharides have been studied from a variety of different aspects, see e.g. Hamada Nobutake: Kobunshi Kako 36:9-16 (1987); and Ohno Naohito: J. Bacteria Japan 527 (2000).
Microbial polysaccharides are formed from different types of monosaccharide units, such as particular types of sugars, including glucose (dextrose, grape sugar) and fructose (fruit sugar). Glucans comprise units of glucose. However, depending on the type of linkage between these subunits, a glucan may be either an α-glucan or β-glucan, Oi et al., Agric. Biol. Chem. 30: 266 (1966).
Microbial polysaccharides may have unique physical properties and physiological activities. For instance, their physical properties can provide consistency, body and substance to foods. They can be utilized as thickening agents and as raw materials in semi-solid foods, such as jellies. Polysaccharides, such as curdlan, are utilized as antioxidant films, Szezesniak et al., J. Food Sci. 27: 381 (1962), Smiey, Food Technol 20: 112 (1966). Moreover, microbial polysaccharides which impart stability, water retention and dispensability may be used in a variety of products, including cosmetics, paints, fertilizers, papermaking, fibers, etc., Jeans, Extracellular Microbiol. Polysaccharides, Sanford et al. (eds), American Chemical Society, Washington, D.C. (1977).
On the other hand, other types of polysaccharides, such as dextran, are used for certain pharmaceutical or medical applications. For instance, dextran is utilized as a plasma substitute or blood flow improver. Polysaccharides, such as chizophyllan, scleroglucan, and curdlan have physiological activities that may be useful in treating diseases such as cancer, Komatsu et al., Gann 60: 137 (1969); Singh et al., Carbohydr. Res. 37: 245 (1974). In general, such polysaccharides do not act directly on cancer cells, but by enhancing immunity. Protein-containing polysaccharides of Trametes versicolor PSK, the polysaccharide lentinan of Lentinus edodes and the polysaccharide schizophyllan of Schizophyllum commune are used as immunotherapeutic drugs for cancer.
An acidic polysaccharide produced by strain FERM P-14228 is described by U.S. Pat. No. 5,789,579. This patent is directed to α-glucans having α-1.4 bonding, unlike the β-glucans of the present invention that have β-1,3-1,6 linkages.
β-glucans are found in mushrooms, such as Agaricus and Lentinus, various starches, cellulose, seaweeds, red algae, yeasts and other similar micro-organisms. Some types of β-glucans play a significant role in health maintenance, see Fujii Noboru: A miracle of β-glucan, Society for Study of Beta Glucan. The present inventors have found that β-1,3-1,6 glucan (Aureobasidium medium) contains much higher quantities of β-glucan than mushrooms—over ten-times more.
Physiological Effects of β-glucans. Many studies have been made with respect to the physiological effects of β-glucans. In particular, the regulatory actions of these compounds on the vital balance and vital immune reaction have been studied. For instance, the protective effects of these compounds in cancer and infectious disease have been studied. Some types of β-glucans, which are of a different type from those of the present invention, have antitumor activities. For instance, these have been observed on 3LL solid tumor cells derived with methylcholanthrene-I in experimental animals, see Fujii Noboru, Shinohara Satoru: Report of the Department of Agriculture, Miyazaki University 33: 243 (1986).
Glucan Receptor. β-glucan receptors are known to be expressed on immunocompetent cells, such as monocytes and macrophages, see Czop et al., J. Exp Med 173: 1511 (1991), Xia et al., J. Immunol. 162: 7285 (1999), Czop, et al., Prog. Clin. Biol. Res. 297: 287 (1989). Certain β-glucans are known to induce the production of inflammatory cytokines from immunocompetent cells, for example, the interleukin 1 receptor antagonist (IL-1Ra) which is an antagonist of interleukin 1β (IL-1β), see Poutsiaka et al., Blood 82: 3695 (1993). It also induces the production of tumor necrosis factorα (TNF-α), interleukin 1β (IL-1β) and platelet activating factor (PAF), and the like, see Abel et al., Int. J. Immunopharmacol. 14: 1363 (1992), Elstad, et al., J. Immunol. 52: 220 (1994).
The molecular structure of the β-glucan receptor has been found to correspond to complement receptor 3 (“CR3”) that binds complement component C3. Recently, this receptor has been shown to be an integrin type CD11b/CD18 molecule itself, see Ross, et al., Complement 4: 61 (1987), Thornton, et al., J. Immunol. 156: 1235 (1996). It has also been found that this CR3 (CD11 b/CD18) molecule is a receptor mainly manifested on the cell surface of monocytes and macrophages and in NK cells. NK cells activated by β-glucan bonding to the CD11b/CD18 molecule via this receptor (CR3) molecule are involved in the destruction and removal of cancer cells, see Di Renzo et al., Eur. J. Immunol. 21: 1755 (1991), Ross, et al., Immunopharmacology 42: 61 (1999), Yan, et al., J. Immunol. 163: 3045 (1999).
Signal transmission. The analysis of signal transmission modes from the β-glucan receptor has been studied. NfkB plays a role as an intranuclear transmission substance of immuno-B-cyte which takes part closely in this signal transmission system, Wakshull et al., Immunopharmacology 41: 89 (1999). Moreover, in model experiments for the therapy of cancer, from the fact that β-glucan is a receptor (CD11b/CD18 molecule) of C3 component of complement, see Yan, et al., J Immunol 163: 3045 (1999), it is reported that the route via this receptor is effective for the destruction of cancer cells opsonized with specific antibody of cancer, see Xia et al., J. Immunol. 162: 2281 (1999).
Drug effects on glucan receptor. On the other hand, functional experiments between drugs having an influence on the immune reaction and β-glucan receptor are also being conducted, and new roles of β-glucan in the vital immune system have become clear in such ways that, in particular, β-glucan receptor (CD11b/CD18 molecule) accelerates its receptor function with glucocorticoid, and the like, see Yashioka et al., FEMS Immunol. Med. Microbiol. 21: 171 (1998), Kay, et al., Immunology 81: 96 (1994).
Glucan effects in vivo. The effects of β-glucans in vivo have also been investigated. Administration of a glucan increases the survival and longevity (macrobiotic effect) of mice exposed to lethal levels of radiation. This effect is attributed to hemopoietic effects of β-glucan and direction of immunocompetent cells into a maturation process, see Patchen et al., Methods Find. Exp. Clin. Pharmacol. 8: 151 (1986). In addition, β-glucans stimulate macrophage function and may thus provide protective effects against certain infectious diseases, such as tuberculosis, see Hetland et al., Scand. J. Immunol 47: 548 (1998). Immunization with the glucan-binding domain of Streptococcus mutans glucosyltransferase provides immunity against infection with Streptococcus mutans, see Jespersgaard et al., Infect. Immun. 67: 6543 (1999).
Immune enhancement with glucans and other substances. While some β-glucans may enhance certain components of the immune system, such as macrophage function, immunological enhancement is a complex phenomenon and at the cell level many intracellular transmission substances are involved in producing these effects. For instance, recently it has been found that mitogen activating protein kinase (MAPK) and calcium controlling the activation and management of immune system are closely involved in these phenomena, see McLeish, et al., J. Leukoc. Biol. 64: 835 (1998), Mork, et al., Immunopharmacology 40: 77 (1998).
β-glucan analogs and diagnosis. Clinically, β-glucan analogues in plasma in patients with pneumonia have been observed. For instance, β-glucan analogues increase in plasma in cases of Pneumocystis carinii pneumonia having no overt AIDS-like symptoms, see Teramoto et al., J. Med. Microbiol. 49: 393 (2000).
While some β-glucans were known to stimulate particular components of the cellular immune system, such as macrophage functions, the present inventors have found that the β-1,3-1,6 glucan (Aureobasidium medium) of the present invention exerts other specific functional activities, such as inducing increases in the numbers of lymphocytes, inducing DNA synthesis and cell division in lymphocytes, exerting direct effects on cancer cells, and directly inhibiting the proliferation of microbes.